Methods for detection of chloral hydrate in dichloroacetic acid

ABSTRACT

Methods for detecting chloral hydrate in dichloroacetic acid are described.

RELATED APPLICATIONS

This application is a continuation of Ser. No. 10/059,776 filed Jan. 29,2002, now U.S. Pat. 6,645,716, which claims benefit to Ser. No.60/264,920 filed Jan. 30, 2001. The contents of each of the foregoingU.S. Patent applications are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

This invention relates generally to novel methods for detecting chloralhydrate in dichoroacetic acid.

BACKGROUND OF THE INVENTION

It is well known that most of the bodily states in multicellularorganisms, including most disease states, are effected by proteins. Suchproteins, either acting directly or through their enzymatic or otherfunctions, contribute in major proportion to many diseases andregulatory functions in animals and man. For disease states, classicaltherapeutics has generally focused upon interactions with such proteinsin efforts to moderate their disease-causing or disease-potentiatingfunctions. In newer therapeutic approaches, modulation of the actualproduction of such proteins is desired. By interfering with theproduction of proteins, the maximum therapeutic effect may be obtainedwith minimal side effects. It is therefore a general object of suchtherapeutic approaches to interfere with or otherwise modulate geneexpression, which would lead to undesired protein formation.

One method for inhibiting specific gene expression is with the use ofoligonucleotides, especially oligonucleotides which are complementary toa specific target messenger RNA (mRNA) sequence. Severaloligonucleotides are undergoing clinic trials for such uses.Oligonucleotides can also serve as competitive inhibitors oftranscription factors, which interact with double-stranded DNA duringregulation of transcription, to modulate their action. Several recentreports describe such interactions (see, for example, Bielinska, A., et.al., Science, 250 (1990), 997–1000; and Wu, H., et. al., Gene, 89,(1990), 203–209).

In addition to such use as both indirect and direct regulators ofproteins, oligonucleotides and their analogs also have found use indiagnostic tests. Such diagnostic tests can be performed usingbiological fluids, tissues, intact cells or isolated cellularcomponents. As with gene expression inhibition, diagnostic applicationsutilize the ability of oligonucleotides and their analogs to hybridizewith a complementary strand of nucleic acid.

Oligonucleotides and their analogs are also widely used as researchreagents. They are useful for understanding the function of many otherbiological molecules as well as in the preparation of other biologicalmolecules. For example, the use of oligonucleotides and their analogs asprimers in PCR reactions has given rise to an expanding commercialindustry. PCR has become a mainstay of commercial and researchlaboratories, and applications of PCR have multiplied. For example, PCRtechnology now finds use in the fields of forensics, paleontology,evolutionary studies and genetic counseling. Commercialization has ledto the development of kits which assist non-molecular biology-trainedpersonnel in applying PCR. Oligonucleotides and their analogs, bothnatural and synthetic, are employed as primers in such PCR technology.

Oligonucleotides and their analogs are also used in other laboratoryprocedures. Several of these uses are described in common laboratorymanuals such as Molecular Cloning, A Laboratory Manual, Second Ed., J.Sambrook, et al., Eds., Cold Spring Harbor Laboratory Press, 1989; andCurrent Protocols In Molecular Biology, F. M. Ausubel, et al., Eds.,Current Publications, 1993. Such uses include as syntheticoligonucleotide probes, in screening expression libraries withantibodies and oligomeric compounds, DNA sequencing, in vitroamplification of DNA by the polymerase chain reaction, and insite-directed mutagenesis of cloned DNA. See Book 2 of MolecularCloning, A Laboratory Manual, supra. See also “DNA-protein interactionsand The Polymerase Chain Reaction” in Vol. 2 of Current Protocols InMolecular Biology, supra. Oligonucleotides and their analogs have alsobeen developed and used in molecular biology in a variety of proceduresas probes, primers, linkers, adapters, and gene fragments.

The widespread use of such oligonucleotides has increased the demand forrapid, inexpensive and efficient procedures for their modification andsynthesis. Early synthetic approaches to oligonucleotide synthesisincluded phosphodiester and phosphotriester chemistries. Khorana et al.,J. Molec. Biol. 72 (1972), 209; Reese, Tetrahedron Lett. 34 (1978),3143–3179. These approaches eventually gave way to more efficient modernmethods, such as the use of the popular phosphoramidite technique (see,e.g., Advances in the Synthesis of Oligonucleotides by thePhosphoramidite Approach, Beaucage, S. L.; Iyer, R. P., Tetrahedron, 48(1992) 2223–2311 and references cited therein), wherein a nucleoside oroligonucleotide having a free hydroxyl group is reacted with a protectedcyanoethyl phosphoramidite monomer in the presence of a weak acid toform a phosphite-linked structure. Oxidation of the phosphite linkagefollowed by hydrolysis of the cyanoethyl group yields the desiredphosphodiester or phosphorothioate linkage.

Solid phase techniques continue to play a large role in oligonucleotidesynthetic approaches. Typically, the 3′-most nucleoside is anchored to asolid support which is functionalized with hydroxyl or amino residues.The additional nucleosides are subsequently added in a step-wise fashionto form the desired linkages between the 3′-functional group of theincoming nucleoside and the 5′-hydroxyl group of the support boundnucleoside. Implicit to this step-wise assembly is use of a protectinggroup to render unreactive the 5′-hydroxy group of the incomingnucleoside. Following coupling, the 5′-hydroxy group is removed throughthe judicious choice of a deprotecting reagent.

Dichloroacetic acid (DCA) is a commonly used reagent for deblockingnucleotides during oligonucleotide synthesis. Because the addition ofnew nucleosides involves the repeated use of dichloroacetic acid fordeprotecting the 5′-hydroxy group, it is important that this reagent beas free as possible of contaminants which may propagate impurities andproduce improper sequences of the target oligonucleotide. Accordingly,methods are needed for detecting such impurities in dichloroacetic acid.The present invention is directed to these, as well as other, importantends.

SUMMARY OF THE INVENTION

It has been discovered that chloral hydrate is a common contaminant incommercially prepared dichloroacetic acid. It has been furtherdiscovered that chloral hydrate reacts with the 5′-hydroxy group ofnucleosides during the course of oligonucleotide synthesis to formundesired side-products that are removed, if at all, only with greatdifficulty. It has been further discovered that chloral hydrate indichloroacetic acid can be detected and its concentration accuratelymeasured, by comparing the integral of a nuclear magnetic resonance peakof the CH proton of chloral hydrate with a known amount of internalstandard.

Accordingly, it is an object of the present invention to provide methodsfor detecting chloral hydrate in dichloroacetic acid.

It is a further object of the present invention to provide methods formeasuring the concentration of chloral hydrate in dichloroacetic acid,particularly, for detecting chloral hydrate and measuring itsconcentration in dichloroacetic acid which is to be used as adeprotecting reagent in oligonucleotide synthesis.

It is a further object of the present invention to provide methods forpreparing oligonucleotides that are free of the impurity which is causedby chloral hydrate present in dichloroacetic acid.

These, as well as other important objects, will be become apparentduring the following detailed description.

DETAILED DESCRIPTION OF THE INVENTION

In a first embodiment, the present invention provides an analyticalmethod comprising determining whether or not a nuclear magneticresonance spectrum taken from a sample of dichloroacetic acid includes anuclear magnetic resonance peak associated with a CH proton of chloralhydrate. In certain preferred embodiments, the method further comprisescomparing an integral of said nuclear magnetic resonance peak associatedwith said CH proton of chloral hydrate with an integral of a nuclearmagnetic resonance peak associated with at least one proton of saiddichloroacetic acid. In certain preferred embodiments, the methodfurther comprises calculating a concentration of chloral hydrate in saiddichloroacetic acid based upon said comparison of integrals of nuclearmagnetic resonance peaks. In certain preferred embodiments, thecalculated concentration of chloral hydrate is less than 15 ppm. Incertain preferred embodiments, the calculated concentration of chloralhydrate is less than 10 ppm. In certain preferred embodiments, thecalculated concentration of chloral hydrate is less than 1 ppm.

In another embodiment, the present invention provides an analyticalmethod comprising determining whether or not a nuclear magneticresonance spectrum taken from a sample of dichloroacetic acid includes anuclear magnetic resonance peak associated with a CH proton of chloralhydrate, further comprising:

adding at least one organic solvent to said sample; and

comparing an integral of said nuclear magnetic resonance peak associatedwith said CH proton of chloral hydrate with an integral of a nuclearmagnetic resonance peak associated with at least one proton of said atleast one organic solvent. In certain preferred embodiments, the organicsolvent is toluene. In certain preferred embodiments, the method furthercomprises calculating a concentration of chloral hydrate in saiddichloroacetic acid based upon said comparison of integrals of nuclearmagnetic resonance peaks. In certain preferred embodiments, thecalculated concentration of chloral hydrate is less than 15 ppm. Incertain preferred embodiments, the calculated concentration of chloralhydrate is less than 10 ppm. In certain preferred embodiments, thecalculated concentration of chloral hydrate is less than 1 ppm. Incertain preferred embodiments, the dichloroacetic acid is used as adeprotecting agent in an oligonucleotide synthesis to prepare anoligonucleotide having the formula:

d(TpCpCpGpTpCpApTpCpGpCpTpCpCpTpCpApGpGpGp) [SEQ ID NO: 1]; whereinP═P(O)S⁻Na⁺.

In another embodiment, the present invention provides an analyticalmethod comprising determining whether or not a nuclear magneticresonance spectrum taken from a sample of dichloroacetic acid includes anuclear magnetic resonance peak associated with a CH proton of chloralhydrate, further comprising contacting an oligonucleotide that bears atleast one protecting group with said dichloroacetic acid. In certainembodiments, contacting effects removal of said at least one protectinggroup from said oligonucleotide. In certain embodiments, saidoligonucleotide from which said protecting group has been removed doesnot include a group having formula 5′-O—CH(OH)(CCl₃). In certainembodiments, said oligonucleotide from which said protecting group hasbeen removed does not include a group having formula 5′-O—CH(CCl₃)—O—.

These, as well as other objects, are a result of the inventors'discovery that chloral hydrate is a common contaminant in commerciallyprepared dichloroacetic acid. As used herein, the term “chloral hydrate”is intended to mean a chemical compound having the structure:Cl₃CH(OH)₂.As used herein, the term “dichloroacetic acid” (DCA) is intended to meana chemical compound having the structure:Cl₂HCC(═O)OH.

It is well-known that dichloroacetic acid (DCA) is a commonly usedreagent for deblocking nucleotides during oligonucleotide synthesis.Particularly, oligonucleotide syntheses involve the repeated use ofdichloroacetic acid as a deprotecting reagent for deblocking 5′-hydroxyprotecting groups (Prot) (Scheme 1):

Thus, as used herein, the term “hydroxyl protecting group” (Prot) isintended to mean a chemical group that is stable under certainconditions but can be removed under other conditions. In general,protecting groups render chemical functionalities inert to specificreaction conditions, and can be appended to and removed from suchfunctionalities in a molecule without substantially damaging theremainder of the molecule. Representative hydroxyl protecting groups aredisclosed by Beaucage, et al., Tetrahedron (1992), 48, 2223–2311, andalso in Greene and Wuts, Protective Groups in Organic Synthesis, Chapter2, 2d ed, John Wiley & Sons, New York, 1991, each of which are herebyincorporated by reference in their entirety. Preferred protecting groupsinclude dimethoxytrityl (DMT), monomethoxytrityl, 9-phenylxanthen-9-yl(Pixyl) and 9-(p-methoxyphenyl)xanthen-9-yl (Mox).

Because of the repetitious use of DCA for the removal of theoligonucleotide protecting groups, it is critical that DCA be free ofcontaminants which may propagate impurities and produce impropersequences of the target oligonucleotide. The inventors have discoveredthat the specific impurity chloral hydrate, when present indichloroacetic acid, reacts to yield side-products, referred to hereinas trichloroethanol adducts 1 and 2, shown below (Scheme 2).

The trichloroethanol adducts are extremely difficult to remove onceformed and, if unchecked, propagate through each synthetic step. Theinventors have discovered that varying degrees of chloral hydrate may bepresent in dichloroacetic acid depending upon the manufacturing processused in its preparation. Thus, a method for detecting the presence andconcentration of chloral hydrate prior to its use in oligonucleotidesynthesis is desirable. Specifications may then be established for themaximum concentrations of chloral hydrate tolerable in anoligonucleotide manufacturing process. In certain embodiments, theconcentration of chloral hydrate tolerated is less than 15 ppm,preferably less than 10 ppm, more preferably less than 1 ppm. Mostpreferably, the concentration of chloral hydrate is below the limit ofdetection.

Accordingly, the present invention is directed, in part, to a method fortesting dichloroacetic acid for the presence of chloral hydrate. Themethod preferably comprises taking a nuclear magnetic resonance (NMR)spectrum of a sample of the dichloroacetic acid prior to its use in amanufacturing process. Preferably, the sample contains a predeterminedconcentration of an internal standard. As used herein, the term“internal standard” or “standard” is intended to mean any compound,preferably a solvent, containing protons, the nuclear magnetic resonancepeak of which may be compared to that of the CH proton of chloralhydrate to determine the concentration of chloral hydrate present. It istherefore preferable that the protons of the internal standard resonateat a location that is different, preferably, upfield or downfield, fromchloral hydrate. Thus, it will be appreciated that in one aspect thepresent invention contemplates using the integral of the peak associatedwith the CH proton of dichloroacetic acid itself as the internalstandard. If the internal standard is other than dichloroacetic acid,the internal standard is preferably selected from suitable liquidsolvents. Such liquid solvents include, but are not limited to,halogenated solvents, hydrocarbon solvents, ether solvents, protic oraprotic solvents.

Suitable halogenated solvents include, but are not limited tobromodichloromethane, dibromochloromethane, bromoform, chloroform,bromochloromethane, dibromomethane, butyl chloride, dichloromethane,trichloroethylene, 1,1,1-trichloroethane, 1,1,2-trichloroethane,1,1-dichloroethane, 2-chloropropane, 1,2,4-trichlorobenzene,o-dichlorobenzene, chlorobenzene, fluorobenzene, dichlorofluoromethane,chlorodifluoromethane, and trifluoromethane.

Suitable hydrocarbon solvents include, but are not limited toacetonitrile, benzene, cyclohexane, pentane, hexane, toluene,cycloheptane, methylcyclohexane, heptane, ethylbenzene, m-, o-, orp-xylene, octane, indane, and nonane.

Suitable ether solvents include, but are not limited todimethoxymethane, tetrahydrofuran, 1,3-dioxane, 1,4-dioxane, furan,diethyl ether, ethylene glycol, dimethyl ether, ethylene glycol diethylether, diethylene glycol dimethyl ether, diethylene glycol diethylether, triethylene glycol diisopropyl ether, anisole, or t-butyl methylether.

Suitable polar protic solvents include, but are not limited to methanol,ethanol, 2-nitroethanol, 2-fluoroethanol, 2,2,2-trifluoroethanol,ethylene glycol, 1-propanol, 2-propanol, 2-methoxyethanol, 1-butanol,2-butanol, i-butyl alcohol, t-butyl alcohol 2-ethoxyethanol, diethyleneglycol, 1-, 2-, or 3-pentanol, neo-pentyl alcohol, t-pentyl alcohol,cyclohexanol, benzyl alcohol, phenol, and glycerol.

Suitable polar aprotic solvents include, but are not limited todimethylformamide (DMF), dimethylacetamide (DMAC),1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU),1,3-dimethyl-2-imidazolidinone (DMI), N-methylpyrrolidinone (NMP),formamide, N-methylacetamide, N-methylformamide, acetonitrile (ACN),dimethylsulfoxide, propionitrile, ethyl formate, methyl acetate,hexachloroacetone, acetone, ethyl methyl ketone, ethyl acetate,isopropyl acetate, t-butyl acetate, sulfolane, N,N-dimethylpropionamide,nitromethane, nitrobenzene, hexamethylphosphoramide.

In preferred embodiments, the internal standard will be stable towardacid. The internal standard is also preferably a solvent which isrelatively non-volatile, thus minimizing the possibility ofconcentration change during standard preparation or NMR acquisition. Inother preferred embodiments, the internal standard is an aproticsolvent. In other preferred embodiments, the internal standard has arelatively simple NMR spectrum, preferably, less than 4 signals. Inparticularly preferred embodiments, the solvent is toluene.

By way of general guidance, a known volume of dichloroacetic acid (DCA)may be dissolved in a known volume of deuterated NMR solvent. If anadditional compound is used as an internal standard, i.e., one that isadded to the NMR tube for the purpose of comparing the integral of thestandard to that of chloral hydrate, it may be added in an concentrationof, for example, approximately 40 ppm. ¹H NMR may then be collectedusing any NMR spectrometer capable of providing conditions suitable foracquiring a resonance signal for each component. A ratio of chloralhydrate to dichloroacetic acid is preferably obtained by comparing theintegral (the area) of the CH proton on chloral hydrate with that of theinternal standard. As will be readily understood by the skilled artisan,the concentration of chloral hydrate in dichloroacetic acid may then becalculated using the ratio obtained via integral comparison, theconcentration of the dichloroacetic acid and the concentration of anyadditional internal standard used. The dichloroacetic acid may then usedaccordingly.

Once the purity of dichloroacetic acid is determined by the methodsdescribed herein, it may subsequently be used for virtually any purpose.Particularly, the dichloroacetic acid may be used in an oligonucleotidesynthesis to produce an oligonucleotide.

As used herein, “oligonucleotide” refers to compounds containing aplurality of monomeric subunits that are joined by phosphorus-containinglinkages, such as phosphite, phosphodiester, phosphorothioate, and/orphosphorodithioate linkages. The monomeric subunits may contain bothnaturally occurring (i.e. “natural”) and non-naturally occurringsynthetic moieties, for example, nucleosidic subunits containingmodified sugar and/or nucleobase portions. Thus, the termoligonucleotide includes oligonucleotides, their analogs, and syntheticoligonucleotides. Such oligonucleotide analogs are typicallystructurally distinguishable from, yet functionally interchangeablewith, naturally occurring or synthetic wild type oligonucleotides. Thus,oligonucleotide analogs include all such structures which functioneffectively to mimic the structure and/or function of a desired RNA orDNA strand, for example, by hybridizing to a target. The term syntheticnucleoside, for the purpose of the present invention, refers to amodified nucleoside. Representative modifications include modificationof a heterocyclic base portion of a nucleoside to give a non-naturallyoccurring nucleobase, a sugar portion of a nucleoside, or bothsimultaneously.

In certain preferred embodiments, the oligonucleotide prepared usingdichloroacetic acid as a deprotecting agent that has been testedaccording to the methods of the present invention has the formula:

d(TpCpCpGpTpCpApTpCpGpCpTpCpCpTpCpApGpGpGp) [SEQ ID NO: 1]; whereinP═P(O)S⁻Na⁺.

As used herein “oligonucleotide synthesis” is intended to have itsart-recognized meaning whereby an oligonucleotide is prepared usingsynthetic methods well known to the ordinarily skilled artisan. By wayof general guidance, the 3′-most nucleoside may be anchored to a solidsupport which is functionalized with hydroxyl or amino residues. Theadditional nucleosides may be subsequently added in a step-wise fashionto form the desired linkages between the 3′-functional group of theincoming nucleoside, and the 5′-hydroxyl group of the support boundnucleoside. In any case, the chosen oligonucleotide synthesis preferablyuses a protecting group to render certain groups unreactive, forexample, the 5′-hydroxy group of the incoming nucleoside. Followingcoupling, the 5′-hydroxy protecting group may then be removed by theaddition of dichloroacetic acid which has been tested for chloralhydrate content.

The present invention is also directed to producing oligonucleotidesthat are free of side products caused by the use of dichloroacetic acidcontaminated with chloral hydrate. One such side product is atrichloroethanol adduct which is the result of chloral hydrate'sreaction with the 5′-hydroxy group (Scheme 2). Thus, as used herein, theterm “trichloroethanol adduct” is intended to mean a nucleoside whereinthe 5′-hydroxy has reacted with chloral hydrate to produce an impurityhaving the moeity: HOCH(CCl₃)—O—CH₂—* wherein * indicates the attachmentpoint to the 5′ position of a nucleoside. It will be appreciated thatfollowing reaction with an incoming nucleoside to form anoligonucleotide, the ethanol adduct will have the structureP—O—CH(CCl₃)—O—CH₂* in an oligonucleotide, wherein P is the phosphorusatom of the incoming nucleoside.

In certain embodiments, the present invention provides a method forproducing an oligonucleotide free of trichloroethanol adduct. Generally,the method comprises testing dichloroacetic acid for the presence ofchloral hydrate prior to use as a deprotecting reagent. If chloralhydrate is present, the concentration can be determined by taking andNMR and comparing the integral of the nuclear magnetic resonance peak ofthe CH proton of chloral hydrate with the integral of the nuclearmagnetic resonance peak of protons of an internal standard to determinethe concentration of chloral hydrate present in the dichloroacetic acid,and using the dichloroacetic acid if the concentration of chloralhydrate is below an acceptable threshold concentration. In certainembodiments, the acceptable concentration of chloral hydrate in thedichloroacetic acid is less than 15 ppm, preferably less than 10 ppm,more preferably 5 ppm, and even more preferably less than 1 ppm. Mostpreferably, the concentration of chloral hydrate is below the level ofdetection.

The oligonucleotides of the present invention may be synthesized throughthe use of a solid support. Solid supports are substrates which arecapable of serving as the solid phase in solid phase syntheticmethodologies, such as those described in Caruthers U.S. Pat. Nos.4,415,732; 4,458,066; 4,500,707; 4,668,777; 4,973,679; and 5,132,418;and Koster U.S. Pat. No. 4,725,677 and U.S. Pat. No. Re. 34,069. Linkersare known in the art as short molecules which serve to connect a solidsupport to functional groups (e.g., hydroxyl groups) of initial synthonmolecules in solid phase synthetic techniques. Suitable linkers aredisclosed in, for example, Oligonucleotides And Analogues A PracticalApproach, Ekstein, F. Ed., IRL Press, N.Y, 1991, Chapter 1, pages 1–23,hereby incorporated by reference in its entirety.

Solid supports according to the invention include those generally knownin the art to be suitable for use in solid phase methodologies,including, for example, controlled pore glass (CPG), oxalyl-controlledpore glass (see, e.g., Alul, et al., Nucleic Acids Research (1991), 19,1527, hereby incorporated by reference in its entirety), TentaGelSupport—an aminopolyethyleneglycol derivatized support (see, e.g.,Wright, et al., Tetrahedron Letters (1993), 34, 3373, herebyincorporated by reference in its entirety) and Poros—a copolymer ofpolystyrene/divinylbenzene.

Following reaction with an incoming nucleoside, the phosphorus atom maybe sulfurized. Sulfurizing agents used during oxidation to formphosphorothioate and phosphorodithioate linkages include Beaucagereagent (see e.g. Iyer, R. P., et. al., J. Chem. Soc. (1990) 112,1253–1254, and Iyer, R. P., et. al., J. Org. Chem. (1990) 55,4693–4699); tetraethylthiuram disulfide (see e.g., Vu, H., Hirschbein,B. L., Tetrahedron Lett. (1991) 32, 3005–3008); dibenzoyl tetrasulfide(see e.g., Rao, M. V., et. al, Tetrahedron Lett. (1992), 33, 4839–4842);di(phenylacetyl)disulfide (see e.g., Kamer, P. C. J., Tetrahedron Lett.,1989, 30, 6757–6760); Bis(O,O-diisopropoxy phosphinothioyl)disulfids(see Stec et al., Tetrahedron Lett., 1993, 34, 5317–5320);3-ethoxy-1,2,4-dithiazoline-5-one (see Nucleic Acids Research, 1996 24,1602–1607, and Nucleic Acids Research (1996) 24, 3643–3644);Bis(p-chlorobenzenesulfonyl)disulfide (see Nucleic Acids Research (1995)23, 4029–4033); sulfur, sulfur in combination with ligands like triaryl,trialkyl, triaralkyl, or trialkaryl phosphines. The foregoing referencesare hereby incorporated by reference in their entirety.

Other useful oxidizing agents used to form the phosphodiester orphosphorothioate linkages include iodine/tetrahydrofuran/water/pyridineor hydrogen peroxide/water or tert-butyl hydroperoxide or any peracidlike m-chloroperbenzoic acid. In the case of sulfurization the reactionis performed under anhydrous conditions with the exclusion of air, inparticular oxygen whereas in the case of oxidation the reaction can beperformed under aqueous conditions.

Oligonucleotides or oligonucleotide analogs prepared by usingdichloroacetic acid tested according to the present invention may behybridizable to a specific target and preferably comprise from about 5to about 50 monomer subunits. It is more preferred that such compoundscomprise from about 10 to about 30 monomer subunits, with 15 to 25monomer subunits being particularly preferred. When used as buildingblocks in assembling larger oligomeric compounds, smaller oligomericcompounds are preferred. Libraries of dimeric, trimeric, or higher ordercompounds can be prepared for use as synthons in the methods of theinvention. The use of small sequences synthesized via solution phasechemistries in automated synthesis of larger oligonucleotides enhancesthe coupling efficiency and the purity of the final oligonucloetides.See, for example: Miura, K., et al., Chem. Pharm. Bull. (1987), 35,833–836; Kumar, G., and Poonian, M. S., J. Org. Chem. (1984) 49,4905–4912; Bannwarth, W., Helvetica Chimica Acta, (1985) 68, 1907–1913;Wolter, A., et al., nucleosides and nucleotides, 1986, 5, 65–77, each ofwhich are hereby incorporated by reference in their entirety.

It will be recognized that the oligonucleotides prepared usingdichloroacetic acid of the invention can be used in diagnostics,therapeutics and as research reagents and kits. They can be used inpharmaceutical compositions by including a suitable pharmaceuticallyacceptable diluent or carrier. The present invention may be furtherunderstood by reference to the following examples.

EXAMPLE 1

Detection of Chloral Hydrate in Dichloroacetic Acid

A test sample of dichloroacetic acid (DCA, 0.15 mL) was dissolved indeuterated acetonitrile (0.5 mL) containing 40 ppm of toluene. ¹H NMRspectra were collected using a Varian Unity 400 NMR spectrometer underthe following conditions: 30 degree pulse, sweep width of 6997.9 Hz, 32k complex points and a 45 second total recycle delay. Approximately 1000transients were run for each sample. The data was processed by zerofilling to 64 k complex points with an exponential line-broadening of0.3 Hz. The first two points were reproduced through linear predictionto smooth base-line and further smoothed after FFT by spline fit.

The concentration of chloral hydrate was measured by comparing theintegral of the peak associate with the CH proton on chloral hydratewith that of the CH₃ group of toluene. Dichloroacetic acid was then usedto prepare the 20 mer according to the procedure set forth in Example 2.

EXAMPLE 2

Preparation of (d(TpCpCpGpTpCpApTpCpGpCpTpCpCpTpCpApGpGpGp) [SEQ ID NO:1]; wherein P═P(O)S⁻Na⁺.

5′-O-DMT-N²-isobutyryl-2′-deoxyguanosine derivatized Primer HL 30support was packed into a steel reactor vessel. A solution ofdichloroacetic acid in toluene (10%, v/v) was added to deprotect theprotected hydroxy group and the product was washed with acetonitrile. Asolution of5′-O-DMT-N²-isobutyryl-2′-deoxyguanosine-3′-O-(2-cyanoethyl)-N,N-diisopropylphosphoramidite(0.2 M) and a solution of 1-H-tetrazole in acetonitrile (0.45 M) wereadded and allowed to react for 5 minutes at room temperature. A solutionof phenylacetyl disulfide in 3-picoline-acetonitrile (0.2 M, 1:1, v/v)was added and allowed to react at room temperature for 2 minutes. Theproduct was washed with acetonitrile (1:4 v/v) andN-methylimidazole-pyridine-acetonitrile (2:3:5, v/v/v). After 2 minutesthe capping mixture was removed by washing the product withacetonitrile.

A solution of dichloroacetic acid in toluene (3% v/v) was added todeprotect the 5′-hydroxy group and the product was washed withacetonitrile. A solution of5′-O-DMT-N²-isobutyryl-2′-deoxyguanosine-3′-O-(2-cyanoethyl)-N,N-diisopropylphosphoramidite(0.2 M) and a solution of 1-H-tetrazole in acetonitrile (0.45 M) wereadded and allowed to react for 5 minutes at room temperature. A solutionof phenylacetyl disulfide in 3-picoline acetonitrile (0.2 M, 1:1, v/v)was added and allowed to react at room temperature for 2 minutes. Theproduct was washed with acetonitrile followed by a capping mixture (1:1,v/v) of acetic anhydride acetonitrile (1:4, v/v) andN-methylimidazole-pyridine-acetonitrile (2:3:5, v/v/v). After 2 minutesthe capping mixture was removed by washing the product withacetonitrile.

The process of deprotecting the 5′-hydroxyl group, adding aphosphoramidite and an activating agent, sufurizing and capping withintervening wash cycles was repeated 17 additional cycles to prepare the20 mer. The resulting support bound oligonucleotide was treated withaqueous ammonium hydroxide (30%) for 12 hours at 60° C. and the productswere filtered. The filtrate was concentrated under reduced pressure anda solution of the residue in water was purified by reversed phase highperformance liquid chromatography. The appropriate fractions werecollected, combined, and concentrated in vacuo. A solution of theresidue in water was treated with aqueous sodium acetate solution (pH3.5) for 45 minutes. The title 2′-deoxyphosphorothioate 20 meroligonucleotide was collected after precipitation by addition ofethanol.

As those skilled in the art will appreciate, numerous changes andmodifications may be made to the preferred embodiments of the inventionwithout departing from the spirit of the invention. It is intended thatall such variations fall within the scope of the invention.

1. A method of preparing an oligonucleotide, comprising: providing asupport-bound nucleotide comprising a 5′-hydroxyl group; coupling anadditional nucleoside to said support-bound nucleoside, said additionalnucleoside comprising a 5′-hydroxy protecting group and a 3′-functionalgroup capable of linking to said 5′-hydroxyl group; selectingdichloroacetic acid having a chloral hydrate concentration of less than15ppm; and removing said 5′-hydroxy protecting group in the presence ofsaid dichloroacetic acid.
 2. The method of claim 1, wherein saiddichloroacetic acid has a chloral hydrate concentration of less than 10ppm.
 3. The method of claim 1, wherein said dichloroacetic acid has achloral hydrate concentration of less than 5 ppm.
 4. The method of claim1, wherein said dichloroacetic has a chloral hydrate concentration ofless than 1 ppm.
 5. The method of claim 1, further comprising taking anuclear magnetic resonance spectrum of said dichloroacetic acid andintegrating a nuclear magnetic resonance peak associated with a CHproton of chloral hydrate.
 6. A method of removing a 5′-hydroxylprotecting group during an oligonucleotide synthesis, comprising:selecting dichloroacetic acid having a chloral hydrate concentration ofless than 15 ppm; and contacting a 5′-hydroxyl protecting group of anoligonucleotide with said dichloroacetic acid.
 7. The method of claim 6,further comprising determining whether a nuclear magnetic resonancespectrum of said dichloroacetic acid includes a nuclear magneticresonance peak associated with a CH proton of chloral hydrate.
 8. Amethod comprising: testing dichloroacetic acid for the presence ofchloral hydrate; determining the chloral hydrate concentration in saiddichloroacetic acid is less than 15 ppm; and contacting a 5′-hydroxyprotecting group of an oligonucleotide with said dichloroacetic acidunder conditions effective to remove said protecting group.
 9. Themethod of claim 8, further comprising coupling a nucleoside to said5′-hydroxy protecting group, said nucleoside comprising a 3′-functionalgroup capable of linking to said 5′-hydroxyl group.